Carbohydrates play a number of important roles in the functioning of living organisms. In addition to their metabolic roles, carbohydrates may be covalently attached to numerous other entities such as proteins and lipids, i.e., glycoconjugates. For example, the carbohydrate portion of glycoproteins may critically affect the ability of glycoproteins to perform their biological functions, including such functions as ligand or receptor recognition.
The enormous potential for chemical and structural diversity among carbohydrates is provided, in part, by the way in which individual sugar units in a polysaccharide can be linked. A fundamental step in determining the three-dimensional structure of a polysaccharide or oligosaccharide is to determine the structure of these linkages. As a consequence of their diverse and important biological functions, aberrations in the synthesis, degradation, or modification of carbohydrates may give rise to altered biologic functions.
Many carbohydrate structures in nature are polysaccharides and oligosaccharides that are produced in a variety of related forms rather than existing in a single defined structure. These families of related carbohydrates are frequently found to be components of the same glycoconjugate. These families of glycoproteins that share the same polypeptide structure, but display variation in the glycosylation pattern have been referred to as glycoforms, Rademacher, et al., Ann. Rev. Biochem., 57:789-838 (1988). Similarly, there is great diversity in the glycoforms associated with glycolipids, proteoglycans, and polysaccharides.
The concentration of individual carbohydrates in a sample may be measured by fluorophore-assisted carbohydrate electrophoresis. The technique of fluorophore-assisted carbohydrate electrophoresis is described in detail in U.S. Pat. No. 4,874,492, U.S. Pat. No. 5,104,508, filed Feb. 14, 1989, and Jackson, et al., Anal. Bioch. 270:705-713 (1990). Fluorophore-assisted carbohydrate electrophoresis permits the electrophoretic separation of a complex mixture of carbohydrates into distinct bands on a gel. Prior to electrophoresis, a carbohydrate mixture for analysis is treated with a charged fluorescent tag that combines with the reducing end of the carbohydrates for analysis. The fluorescent label permits the quantitative measurement of the labelled carbohydrates. The charged tag not only fluorescently labels the carbohydrates, but imparts an ionic charge, thus permitting hitherto uncharged carbohydrates to migrate in an electric field. After the carbohydrates have been labelled, the sample is subjected to polyacrylamide gel electrophoresis in order to separate and concentrate the labelled carbohydrates into bands. The separated carbohydrates may be visualized directly by fluorescence under U.V. light. Alternatively the separated carbohydrates may be visualized by means of laser-scanner photomultiplier tube system, a charge coupled device (CCD). CCD's are semiconductor imaging devices that permit the sensitive detection of emitted light. CCD's and their uses are described in U.S. Pat. Nos. 4,874,492 and 4,892,137. The image produced by the CCD may be subsequently transferred to a computer wherein the bands may be analyzed with the respect to parameters such as intensity, mobility, migration distance, and the like.
To date only a relatively small percentage of the carbohydrate-interacting protein, i.e., carbohydrate-modifying enzyme and carbohydrate-interacting protein, genes thought to exist in nature have been isolated. A principal reason for the limited number of successfully cloned carbohydrate-interacting protein genes is the lack of sensitive and specific assays for the identification of clones of interest during the screening of genetic libraries. Traditional methods for the cloning of these enzymes requires the use of monoclonal antibodies, polyclonal antibodies, or oligonucleotide hybridization probes based on protein sequence information for the screening of genetic libraries.
Thus, it is of interest to provide highly sensitive and highly specific assays for detecting and/or quantitating carbohydrate-interacting protein activity. Such highly specific and highly sensitive assays would have a number of uses that are difficult, or impossible, to achieve using currently available carbohydrate-interacting protein assay technology. These uses include the purification, detection and discovery of carbohydrate-interacting proteins having novel specificities. Highly specific and sensitive assays also find use in the cloning of genes encoding carbohydrate-interacting proteins.